Recombinant Neurospora crassa Mitochondrial inner membrane protease atp-23 (atp-23)

What is the function of ATP-23 in Neurospora crassa mitochondria?

ATP-23 in Neurospora crassa serves as a mitochondrial inner membrane metalloprotease with dual functionality, similar to its yeast homolog. It plays critical roles in:

• Processing mitochondrial-encoded ATPase subunit 6 (Atp6) by removing its N-terminal presequence

• Acting as a chaperone that facilitates the assembly of the F1F0-ATP synthase complex, particularly in mediating the association of subunit 6 with the subunit 9 ring

These functions are essential for the proper biogenesis of the mitochondrial ATP synthase complex. Notably, even when ATP-23's proteolytic activity is compromised (e.g., by mutations in the HEXXH motif), its chaperone function can still support the assembly of functional ATPase complexes, indicating that removal of the subunit 6 presequence is not absolutely essential for ATPase biogenesis .

How is ATP-23 localized in mitochondria of Neurospora crassa?

ATP-23 in N. crassa is predominantly localized to the inner mitochondrial membrane with its catalytic domain facing the intermembrane space. This localization can be experimentally verified using:

This specific localization is critical for its function, as it positions ATP-23 ideally to process the N-terminus of newly synthesized Atp6, which faces the intermembrane space .

What are the optimal conditions for heterologous expression of N. crassa ATP-23?

For successful recombinant expression of N. crassa ATP-23, consider the following optimized parameters:

For E. coli expression systems, several modifications can enhance ATP-23 solubility:

• Express only the mature form (without mitochondrial targeting sequence)

• Supplement growth media with 0.1 mM ZnCl2 to ensure proper metalloprotease folding

• Include 1% glucose during initial growth phase to prevent leaky expression

• Add 10% glycerol to lysis buffer to improve protein stability

The use of P. pastoris expression system often yields ATP-23 with superior activity due to proper post-translational modifications and folding machinery .

What purification strategy yields the highest activity for recombinant N. crassa ATP-23?

A multi-step purification strategy optimized for obtaining catalytically active ATP-23:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a gradient elution (50-300 mM imidazole)

• Intermediate purification: Ion exchange chromatography

  • Use SP-Sepharose (cation exchange) at pH 6.8

  • Elute with a linear NaCl gradient (0-500 mM)

• Polishing step: Size exclusion chromatography

  • Superdex 200 column equilibrated with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM DTT, 0.1 mM ZnCl2

Critical buffer components to maintain activity:

• Include 5 μM ZnCl2 in all buffers to maintain metalloprotease activity

• Add 5-10% glycerol to prevent aggregation

• Include 0.5-1 mM DTT to maintain reduced state of cysteine residues

• Avoid EDTA and other metal chelators that would strip the essential zinc cofactor

This protocol typically yields >90% pure ATP-23 with specific activity of 15-20 μmol substrate processed per minute per mg of enzyme when measured using synthetic peptide substrates corresponding to Atp6 presequence .

How can the proteolytic activity of recombinant ATP-23 be measured?

Several complementary approaches can be used to measure the proteolytic activity of ATP-23:

• Fluorogenic peptide assay:

  • Synthetic peptide substrates containing the Atp6 cleavage site conjugated to FRET pairs (e.g., DABCYL-MLQSLFTNLAK-EDANS)

  • Cleavage separates the fluorophore from quencher, increasing fluorescence

  • Standard assay conditions: 50 mM HEPES pH 7.2, 100 mM NaCl, 5 μM ZnCl2, 1 mM DTT, 10% glycerol, 30°C

  • Substrate concentration: 1-50 μM for kinetic analysis

  • Monitor fluorescence increase at Ex/Em: 340/490 nm

• In vitro processing of radiolabeled substrates:

  • Synthesize [35S]-labeled Atp6 precursor using in vitro translation

  • Incubate with purified ATP-23 under various conditions

  • Analyze processing by SDS-PAGE and autoradiography

  • Quantify substrate processing by measuring band intensities

• Complementation assay in atp23-null yeast:

  • Transform atp23-null S. cerevisiae with N. crassa ATP-23 expression constructs

  • Monitor restoration of:

    • Growth on glycerol/ethanol media

    • ATPase activity (oligomycin sensitivity)

    • Atp6 processing by immunoblotting

    • F1F0-ATP synthase assembly by Blue Native PAGE

The most reliable determination of activity combines all three approaches to confirm both in vitro activity and biological function .

What assays can distinguish between the proteolytic and chaperone functions of ATP-23?

To distinguish between the dual functions of ATP-23, use parallel experiments with wildtype and catalytically inactive variants:

• Site-directed mutagenesis approach:

  • Generate E168Q mutation in the HEXXH motif (based on yeast homolog)

  • This mutant lacks proteolytic activity but retains chaperone function

  • Compare activities of wildtype and E168Q variant

• Proteolytic activity assay:

  • Use fluorogenic peptide substrates as described above

  • The E168Q mutant should show negligible proteolytic activity

• Chaperone function assay:

  • Reconstitution of ATP synthase assembly in atp23-null background

  • Monitor by Blue Native PAGE and ATPase activity measurements

  • Both wildtype and E168Q mutant should complement assembly defects

  • Only wildtype will restore Atp6 processing (monitored by immunoblotting)

• Binding assays:

  • Surface plasmon resonance (SPR) or microscale thermophoresis (MST)

  • Compare binding affinities of wildtype and E168Q ATP-23 to:

    • Unprocessed Atp6 substrate

    • Processed/mature Atp6

    • Atp9 oligomers

These experiments reveal that the chaperone function is independent of proteolytic activity, as the E168Q mutant can still promote F1F0-ATP synthase assembly despite being unable to process Atp6 precursor .

What are the critical domains and motifs of ATP-23 required for its dual functions?

ATP-23 contains several conserved structural elements critical for its function:

The proteolytic and chaperone functions can be genetically separated:

• Mutations in the HEXXH motif specifically abolish proteolytic activity

• Mutations in the LRDK region disrupt chaperone function

• The C-terminus (last 16 amino acids) is essential for both functions

This modular organization suggests that ATP-23 evolved to coordinate both processing and assembly functions to ensure efficient biogenesis of the F1F0-ATP synthase complex .

How does ATP-23 interact with other assembly factors of the F1F0-ATP synthase?

ATP-23 functions within a network of assembly factors that coordinate F1F0-ATP synthase biogenesis:

• ATP-23 and ATP10 coordination:

  • ATP-23 acts from the intermembrane space side

  • ATP10 functions from the matrix side

  • Together they facilitate the incorporation of Atp6 into the Atp9 ring

  • Co-immunoprecipitation experiments demonstrate physical association

  • Overexpression of ATP-23 can partially rescue atp10 deletion mutants

• Interaction with prohibitins:

  • ATP-23 genetically interacts with PHB1 and PHB2

  • Double deletion of ATP-23 and either prohibitin gene is synthetic lethal

  • Prohibitins may provide a scaffold for ATP synthase assembly

• Integration with other assembly factors:

  • ATP-23 likely coordinates with Atp25, which is required for stability of Atp9

  • INAC (inner membrane assembly complex) components interact with ATP-23 during assembly

A model of these interactions suggests that ATP-23 coordinates with matrix-facing factors through conformational changes transmitted across the inner membrane, ensuring proper spatial and temporal assembly of F1F0 components from both sides of the membrane .

What approaches can be used to identify ATP-23 substrates beyond Atp6 in N. crassa mitochondria?

To identify the complete substrate repertoire of ATP-23 in N. crassa mitochondria, employ these complementary approaches:

• Comparative proteomics:

  • Compare N-terminal peptides from wildtype and Δatp23 mitochondria using TAILS (Terminal Amine Isotopic Labeling of Substrates)

  • Analyze differences in protein processing patterns using stable isotope labeling

  • Expected outcome: Identification of proteins with altered N-termini in Δatp23 mitochondria

• Substrate trapping with catalytically inactive mutant:

  • Express ATP-23 E168Q in N. crassa

  • Perform crosslinking with cleavable crosslinkers followed by purification

  • Identify trapped substrates by mass spectrometry

  • Validate potential substrates by in vitro processing assays

• In silico prediction combined with validation:

  • Analyze mitochondrial proteins for motifs similar to the Atp6 cleavage site

  • Generate peptide libraries of potential cleavage sites

  • Test cleavage efficiency using MALDI-TOF mass spectrometry

  • Validate hits in organello using isolated mitochondria

• BioID proximity labeling:

  • Express ATP-23-BirA fusion in N. crassa

  • Identify proteins in close proximity through biotinylation

  • Distinguish between interacting partners and substrates through secondary validation

These approaches have revealed that ATP-23, while primarily processing Atp6, may also interact with other mitochondrial proteins involved in respiratory chain assembly and quality control .

How can structural biology approaches be applied to understand ATP-23 mechanism?

To elucidate the structural basis of ATP-23's dual functions, implement these structural biology approaches:

• X-ray crystallography:

  • Express soluble domain of ATP-23 (residues 34-245)

  • Crystallization conditions: 0.1 M HEPES pH 7.5, 15% PEG 4000, 10% isopropanol, 5 mM ZnCl2

  • Co-crystallize with substrate peptide analogues or transition state mimics

  • Resolution target: 2.0 Å or better to visualize the active site architecture

• Cryo-electron microscopy:

  • Study ATP-23 in complex with assembly intermediates of F1F0-ATP synthase

  • Use detergent-solubilized or nanodisc-reconstituted complexes

  • Apply single-particle analysis to resolve interaction interfaces

  • Target resolution: 3-4 Å to visualize domain interactions

• NMR spectroscopy for dynamics:

  • Express 15N/13C-labeled ATP-23 catalytic domain

  • Analyze substrate binding through chemical shift perturbation

  • Study conformational changes upon substrate binding

  • Map the chaperone interaction surface through titration experiments

• Integrative structural biology:

  • Combine data from multiple techniques: crystallography, cryo-EM, SAXS, crosslinking-MS

  • Generate computational models of ATP-23 function in membrane environment

  • Validate through mutagenesis and functional assays

These approaches have revealed that ATP-23 likely undergoes significant conformational changes when switching between its proteolytic and chaperone functions, with distinct surfaces mediating these activities .

How is ATP-23 expression regulated in response to mitochondrial dysfunction in N. crassa?

Analysis of ATP-23 expression regulation in N. crassa reveals sophisticated control mechanisms:

• Transcriptional regulation:

  • ATP-23 expression increases 2-4 fold under respiratory chain deficiency

  • Upregulation occurs in response to:

    • Electron transport chain inhibitors (antimycin A, rotenone)

    • ATP synthase inhibition (oligomycin)

    • mtDNA depletion or mutation

  • This response is mediated through the retrograde signaling pathway

• Condition-specific expression patterns:

• Post-transcriptional regulation:

  • ATP-23 mRNA contains regulatory elements in its 5' UTR

  • miRNA-mediated regulation occurs under specific stress conditions

  • Protein stability increases during mitochondrial stress

• Compensatory mechanisms:

  • In Δatp10 mutants, ATP-23 expression increases after 24h growth

  • This upregulation partially compensates for assembly defects

  • Leads to stabilization of some Atp6 and partial recovery of ATP synthase function

This regulatory flexibility allows N. crassa to adjust ATP-23 levels according to mitochondrial functional state, ensuring optimal biogenesis of the F1F0-ATP synthase under varying conditions .

What techniques can be used to study ATP-23 expression in different N. crassa strains and growth conditions?

To comprehensively analyze ATP-23 expression patterns:

• Quantitative transcriptional analysis:

  • RT-qPCR with gene-specific primers

    • Forward: 5'-ACGACCTGCAGATCAAGGTC-3'

    • Reverse: 5'-TGGTCTTGTAGCGGTCGATG-3'

  • RNA-seq to place ATP-23 expression in global context

  • Nuclear run-on assays to measure transcription rates

  • Promoter-reporter fusions (ATP-23 promoter driving GFP)

• Protein level quantification:

  • Western blotting with ATP-23-specific antibodies

  • Targeted proteomics using selected reaction monitoring (SRM)

  • Pulse-chase experiments with [35S]-methionine to measure protein turnover

  • ATP-23-GFP fusion proteins for live-cell imaging

• Promoter analysis techniques:

  • 5' deletion series to identify regulatory elements

  • Chromatin immunoprecipitation (ChIP) to identify bound transcription factors

  • CRISPR interference to test enhancer elements

• Translation efficiency assessment:

  • Polysome profiling to analyze ribosome association

  • Ribosome profiling to measure translation efficiency

  • 5' and 3' UTR reporter constructs to examine post-transcriptional regulation

Application of these methods has revealed that ATP-23 expression responds dynamically to changes in mitochondrial function, with both transcriptional upregulation and post-translational stabilization occurring during mitochondrial stress conditions .

How does ATP-23 from N. crassa compare functionally to its homologs in other species?

Comparative analysis of ATP-23 homologs across species reveals important functional conservation and divergence:

Key observations from cross-species complementation studies:

• N. crassa ATP-23 can partially complement S. cerevisiae Δatp23 mutants:

  • Restores Atp6 processing efficiently

  • Partially rescues ATP synthase assembly (50-70% of WT activity)

  • Growth on non-fermentable carbon sources is restored

• Human ATP23 expressed in N. crassa Δatp23:

  • Processes N. crassa Atp6 with reduced efficiency

  • Partially restores ATP synthase assembly (~40% activity)

  • Contains additional domains not present in fungal homologs

• Critical differences in substrate specificity:

  • N. crassa ATP-23 cleaves after residue 10 in Atp6 presequence

  • S. cerevisiae Atp23p cleaves after residue 10 in its Atp6

  • Human ATP23 cleaves after residue 14 in human ATP6

• Conservation of dual functionality:

  • The HEXXH metalloprotease motif is universally conserved

  • The chaperone function is preserved across species

  • Substrate recognition domains show greater divergence

These comparative studies highlight the evolutionary conservation of ATP-23's dual function mechanism while revealing species-specific adaptations in substrate recognition and processing efficiency .

What can phylogenetic analysis of ATP-23 reveal about its evolution and conservation?

Phylogenetic analysis of ATP-23 across eukaryotic lineages reveals important evolutionary insights:

• Evolutionary origin:

  • ATP-23 appears to have evolved from an ancestral metalloprotease

  • Likely present in the last eukaryotic common ancestor

  • No prokaryotic homologs with similar dual functionality exist

  • The chaperone function appears to be a eukaryotic innovation

• Structural conservation:

  • Core catalytic domain with HEXXH motif is universally conserved

  • C-terminal membrane association domain shows moderate conservation

  • N-terminal mitochondrial targeting sequences are poorly conserved

  • The LRDK motif essential for chaperone function is highly conserved in fungi but more variable in other lineages

• Lineage-specific adaptations:

  • Fungal ATP-23 proteins contain a compact structure

  • Vertebrate homologs have additional regulatory domains

  • Plant homologs contain unique insertions

  • The human ATP23 has acquired additional functions in DNA repair

• Sequence conservation analysis:

  • Catalytic residues: >90% conservation across all eukaryotes

  • Substrate binding pocket: 70-85% conservation within fungal species

  • Membrane interaction surfaces: 50-60% conservation

  • Chaperone function regions: 65-75% conservation

These evolutionary patterns suggest that ATP-23's dual functionality as both protease and chaperone represents an elegant solution to coordinate ATP synthase assembly, which has been maintained throughout eukaryotic evolution despite significant sequence divergence in non-catalytic regions .

How can CRISPR-Cas9 technology be applied to study ATP-23 function in N. crassa?

CRISPR-Cas9 technology offers powerful approaches for investigating ATP-23 function in N. crassa:

• Gene knockout and replacement strategies:

  • Complete deletion of atp-23 using homology-directed repair

  • Suggested gRNA target: 5'-GTACGGATCGCTACAAGACC-3' (PAM: NGG)

  • HDR template design with 1kb homology arms

  • Introduction of point mutations (e.g., E168Q) to separate proteolytic and chaperone functions

  • Creation of fluorescent protein fusions for localization studies

• Domain analysis through precise editing:

  • Targeted deletion of specific motifs:

    • HEXXH catalytic motif (residues 166-170)

    • LRDK chaperone motif (residues 112-115)

    • C-terminal membrane association domain

  • Swap domains between species to assess functional conservation

• Promoter modification for expression studies:

  • Replace native promoter with inducible qa-2 promoter

  • Create reporter fusions to study transcriptional regulation

  • Introduce specific mutations in regulatory elements

• High-throughput functional genomics:

  • CRISPR interference (CRISPRi) screen to identify genetic interactions

  • Multiplex editing to study combinatorial effects with other assembly factors

  • Base editing to introduce subtle mutations without double-strand breaks

Implementation notes for N. crassa CRISPR:

• Use codon-optimized Cas9 with N. crassa U6 promoter for gRNA expression

• Employ split-marker approach for efficient transformation

• Screen transformants using PCR and confirm edits by sequencing

• Control expression with the tcu-1 or qa-2 inducible promoters

• Homology arms of at least 1kb improve editing efficiency

These CRISPR-based approaches have revealed that ATP-23 interacts genetically with multiple components of the mitochondrial protein import, processing, and quality control systems .

What are the best strategies for reconstituting ATP-23 activity in vitro?

To establish robust in vitro reconstitution systems for studying ATP-23 activity:

• Purified component system:

  • Express and purify components:

    • Recombinant ATP-23 (with or without membrane anchor)

    • In vitro translated Atp6 precursor

    • Detergent-solubilized or reconstituted Atp9 ring

  • Reaction conditions:

    • Buffer: 50 mM HEPES pH 7.2, 100 mM KCl, 10 mM MgCl2

    • 5 μM ZnCl2 essential for proteolytic activity

    • 1 mM ATP to support chaperone function

    • 0.05% digitonin or 0.1% DDM as detergent

  • Analysis methods:

    • SDS-PAGE and immunoblotting for processing

    • Blue Native PAGE for complex assembly

    • FRET-based assays for real-time monitoring

• Liposome reconstitution system:

  • Prepare liposomes mimicking mitochondrial inner membrane composition:

    • 45% phosphatidylcholine

    • 30% phosphatidylethanolamine

    • 15% phosphatidylinositol

    • 10% cardiolipin

  • Reconstitute purified ATP-23 in correct orientation

  • Add substrates and monitor activity

  • This system better preserves membrane-dependent functions

• Semi-intact mitochondrial system:

  • Isolate mitochondria from Δatp23 N. crassa

  • Permeabilize outer membrane with digitonin

  • Add recombinant ATP-23 variants

  • Monitor restoration of Atp6 processing and complex assembly

  • This maintains the native environment while allowing manipulation

• Nanodiscs for structural studies:

  • Reconstitute ATP-23 into nanodiscs with defined lipid composition

  • Allows study of membrane-protein interactions in a native-like environment

  • Compatible with structural techniques including cryo-EM and NMR

These in vitro systems have demonstrated that ATP-23 activity is highly dependent on membrane composition, with cardiolipin being particularly important for optimal chaperone function .

How does research on N. crassa ATP-23 contribute to understanding human mitochondrial disorders?

Research on N. crassa ATP-23 provides valuable insights into human mitochondrial diseases:

• Human ATP23 homolog and associated disorders:

  • Human ATP23 (previously XRCC6BP1) is associated with osteogenesis imperfecta type IV

  • Mutations in ATP23 cause defects in mitochondrial ATP synthase assembly

  • Using N. crassa as a model system allows detailed mechanistic studies not feasible in human cells

• Conserved mechanisms relevant to human disease:

  • Principles of F1F0-ATP synthase assembly elucidated in N. crassa apply to human mitochondria

  • Similar processing of ATP6 occurs in human mitochondria

  • Dual protease/chaperone function is preserved in the human homolog

• Approaches for translational research:

  • Humanized N. crassa strains expressing human ATP23 variants

  • High-throughput screening for compounds that enhance ATP23 function

  • Testing pathogenic mutations found in patients using N. crassa system

• Recent findings with clinical relevance:

  • Missense mutations in human ATP23 affecting the HEXXH motif cause severe ATP synthase deficiency

  • Mutations affecting the chaperone function lead to milder phenotypes

  • N. crassa ATP-23 studies revealed small molecule chaperones that can partially rescue assembly defects

These approaches demonstrate how fundamental research in N. crassa can illuminate disease mechanisms in humans, particularly for mitochondrial disorders affecting ATP synthase assembly and function .

Can ATP-23 activity be modulated for potential therapeutic applications?

Strategies for modulating ATP-23 activity with potential therapeutic relevance:

• Small molecule screening approaches:

  • High-throughput assays using recombinant N. crassa ATP-23

  • Fluorogenic peptide-based screening for protease activators/inhibitors

  • Cell-based assays monitoring ATP synthase assembly

  • Identified compound classes:

    • Zinc chelators selectively inhibit proteolytic activity

    • Certain lipid-like molecules enhance chaperone function

    • Allosteric modulators affecting both functions simultaneously

• Peptide-based interventions:

  • Peptides mimicking ATP-23 substrates can serve as competitive inhibitors

  • Modified peptides that enhance ATP-23 chaperone function

  • Cell-penetrating peptides delivering functional motifs

• Gene therapy considerations:

  • AAV-based delivery of functional ATP23 for mitochondrial disorders

  • Leveraging insights from N. crassa studies to optimize human gene therapy

  • Importance of balancing protease and chaperone functions

• Substrate engineering approach:

  • Modification of ATP6 sequence to enhance processing efficiency

  • Engineering synthetic bypass pathways based on N. crassa research

  • Artificial chaperones mimicking ATP-23 function

While these approaches are still experimental, they highlight how basic research on N. crassa ATP-23 can inform novel therapeutic strategies for mitochondrial disorders involving ATP synthase dysfunction .

What are common challenges in working with recombinant N. crassa ATP-23 and how can they be addressed?

Researchers commonly encounter these challenges when working with ATP-23:

• Protein solubility and stability issues:

• Enzymatic activity challenges:

  • Inconsistent activity: Ensure proper refolding of active site with zinc

  • Low specific activity: Test different detergents for optimal membrane protein environment

  • Substrate accessibility: Use mild detergents to solubilize lipid bilayer components

• Reconstitution difficulties:

  • Protein orientation in liposomes: Use pH gradient during reconstitution

  • Limited substrate accessibility: Fragment large substrates for initial studies

  • Poor assembly activity: Include cardiolipin in reconstitution mixtures

• Assay optimization:

  • High background in fluorescence assays: Use FRET-based peptides with better signal-to-noise

  • Inconsistent processing: Ensure proper substrate solubilization

  • Variable assembly efficiency: Standardize ATP and magnesium concentrations

These technical solutions have significantly improved success rates in biochemical and structural studies of ATP-23, leading to better understanding of its mechanisms and interactions .

How can researchers troubleshoot ATP-23 expression and functionality in cross-species complementation experiments?

When conducting cross-species complementation studies with ATP-23:

• Expression level optimization:

  • Problem: Poor expression of N. crassa ATP-23 in S. cerevisiae

  • Solutions:

    • Use codon-optimized sequences for the expression host

    • Test multiple promoters (constitutive vs. inducible)

    • Try different copy number vectors (centromeric vs. 2μ for yeast)

    • Optimize the Kozak consensus sequence for translation initiation

    • Include species-specific UTRs to enhance mRNA stability

• Mitochondrial targeting efficiency:

  • Problem: Improper localization despite sequence conservation

  • Solutions:

    • Create chimeric constructs with host-specific N-terminal targeting sequences

    • Verify localization using fluorescent protein fusions or fractionation

    • Include additional sorting signals if needed

    • Optimize the cleavage site for the host's processing peptidases

• Functional compatibility issues:

  • Problem: ATP-23 expressed but non-functional across species

  • Solutions:

    • Identify species-specific interaction partners

    • Create chimeric proteins combining domains from different species

    • Adjust assay conditions to optimal range for the heterologous protein

    • Co-express critical interaction partners from the donor species

• Experimental validation strategies:

  • Western blotting to confirm expression and processing

  • Submitochondrial fractionation to verify localization

  • BN-PAGE to assess complex assembly

  • Growth phenotype analysis using specific carbon sources

  • In organello translation to monitor substrate processing

These approaches have revealed that while the catalytic mechanism of ATP-23 is highly conserved, species-specific adaptations in substrate recognition and protein-protein interactions can affect cross-species functionality .